Tissue-type plasminogen activator (tPA) is the approved therapy for acute thrombotic stroke. tPA promotes intravascular fibrinolysis, but increases risk for hemorrhage and is neurotoxic. Activated protein C (APC) has anti-thrombotic activity and is neuroprotective in a mouse model of stroke. In human hypoxic brain endothelium and in mice in vivo, we have shown that APC activates anti-apoptotic pathway through endothelial protein C receptor (EPCR)-dependent activation of protease activated receptor 1 (PAR1) and blockade of p53-dependent apoptosis. Our preliminary data suggest that APC acting via PAR1 and PAR3 directly protects mouse cortical cells from N-methyl-D-aspartate (NMDA)-induced apoptosis by blocking p53 and caspase-3 signaling and that APC limits NMDA excitotoxicity in vivo. Our central hypothesis is that APC and certain of its functional mutants protect the brain from ischemic/thrombotic events after stroke by combined anti-thrombotic activity and direct anti-apoptic effects on brain cells through PAR1, PAR3 and EPCR by acting on both neurons and brain endothelium. APC/tPA combined therapy for stroke should exert synergistic neuroprotective effects through the combination of anti-thrombotic and fibrinolytic intravascular activities of APC and tPA, respectively, while APC should limit tPA's direct neurotoxicity by activating anti-apoptotic mechanisms in perturbed brain cells. Recombinant human and mouse tPA and mouse wild-type and mutant APCs with reduced anticoagulant activity but normal cytoprotective activity (termed "APC functional mutants") will be studied. The research design proposes to test the hypothesis, first, in an in vivo mouse model of focal ischemic stroke with secondary brain microvascular thrombosis (aim 1); second, in an in vivo mouse model of NMDA-induced and tPA-induced excitotoxic brain injury to isolate APC/tPA systemic effects from their direct effects on brain cells (aim 2); and third, in an in vitro cell culture model of NMDA induced apoptosis in mouse cortical neurons (aim 3). We will study PAR1, PAR3 and PAR4 null mice and severely EPCR-deficient mice. Studies proposed in this application will help to determine the safety and efficacy of new APC/tPA combined therapies for thrombotic stroke and will provide new mechanistic insights into the anti-thrombotic and anti-apoptotic activities of APC and its functional mutants.